A Patient with Pontocerebellar Hypoplasia Type 6: Novel RARS2 Mutations, Comparison to Previously Published Patients and Clinical Distinction from PEHO Syndrome

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A patient with pontocerebellar hypoplasia type 6: Novel RARS2 mutations, comparison to previously published patients and clinical distinction from PEHO syndrome

Viivi Nevanlinna, Svetlana Konovalova, Berten Ceulemans, Mikko Muona, Anni Laari, Taru Hilander, Katarin Gorski, Leena Valanne, Anna-Kaisa Anttonen, Henna Tyynismaa, Carolina Courage, Anna-Elina Lehesjoki PII: S1769-7212(18)30943-1 DOI: https://doi.org/10.1016/j.ejmg.2019.103766 Reference: EJMG 103766

To appear in: European Journal of Medical Genetics

Received Date: 21 December 2018 Revised Date: 15 September 2019 Accepted Date: 15 September 2019

Please cite this article as: V. Nevanlinna, S. Konovalova, B. Ceulemans, M. Muona, A. Laari, T. Hilander, K. Gorski, L. Valanne, A.-K. Anttonen, H. Tyynismaa, C. Courage, A.-E. Lehesjoki, A patient with pontocerebellar hypoplasia type 6: Novel RARS2 mutations, comparison to previously published patients and clinical distinction from PEHO syndrome, European Journal of Medical Genetics (2019), doi: https://doi.org/10.1016/j.ejmg.2019.103766.

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1 A Patient with Pontocerebellar Hypoplasia Type 6: Novel RARS2 Mutations, Comparison to

2 Previously Published Patients and Clinical Distinction from PEHO Syndrome

4 Viivi Nevanlinna a,b , Svetlana Konovalova c, Berten Ceulemans d, Mikko Muona a,1, Anni Laari a,c , Taru

5 Hilander c, Katarin Gorski a,c , Leena Valanne e, Anna-Kaisa Anttonen a,f,g, Henna Tyynismaa c, f, h ,

6 Carolina Courage a,c , Anna-Elina Lehesjoki a,c,f,*

8 aFolkhälsan Research Center, Helsinki, Finland

9 bFaculty of Medicine and Life Sciences, University of Tampere, Tampere, Finland

10 cResearch Programs Unit, Molecular Neurology, University of Helsinki, Finland

11 dDepartment of Pediatric Neurology, Antwerp University Hospital, University of Antwerp, Belgium

12 eDepartment of Radiology, Hospital District of Helsinki and Uusimaa Medical Imaging Center,

13 University of Helsinki and Helsinki University Hospital, Helsinki, Finland

14 fDepartment of Medical and Clinical Genetics, Medicum, University of Helsinki, Finland

15 gLaboratory of Genetics, Helsinki University Hospital, Helsinki, Finland

16 hNeuroscience Center, HiLIFE, University of Helsinki, Helsinki, Finland

17 1Present address: Blueprint Genetics, Helsinki, Finland

18 *Corresponding author. Folkhälsan Research Center, Biomedicum Helsinki, Haartmaninkatu 8,

19 00290, Helsinki, Finland; tel: +358 2941 25072; email: [email protected]

20 Abstract

21 Pontocerebellar hypoplasia type 6 (PCH6) is a rare infantile-onset progressive encephalopathy

22 caused by biallelic mutations in RARS2 that encodes the mitochondrial arginine-tRNA synthetase

23 enzyme (mtArgRS). The clinical presentation overlaps that of PEHO syndrome (Progressive

24 Encephalopathy with oedema, Hypsarrhythmia and Optic atrophy). The proband presented with

25 severe intellectual disability, epilepsy with varying seizure types, optic atrophy, axial hypotonia,

26 acquired microcephaly, dysmorphic features and progressive cerebral and cerebellar atrophy and

27 delayed myelination on MRI. The presentation had resemblance to PEHO syndrome but

28 sequencing of ZNHIT3 did not identify pathogenic variants. Subsequent whole genome sequencing

29 revealed novel compound heterozygous variants in RARS2 , a missense variant affecting a highly

30 conserved amino acid and a frameshift variant with consequent degradation of the transcript

31 resulting in decreased mtArgRS protein level confirming the diagnosis of PCH6. Features

32 distinguishing the proband’s phenotype from PEHO syndrome were later appearance of hypotonia

33 and elevated lactate levels in blood and cerebrospinal fluid. On MRI the proband presented with

34 more severe supratentorial atrophy and lesser degree of abnormal myelination than PEHO

35 syndrome patients. The study highlights the challenges in clinical diagnosis of patients with

36 neonatal and early infantile encephalopathies with overlapping clinical features and brain MRI

37 findings.

39 Keywords

40 Pontocerebellar hypoplasia type 6, RARS2, PEHO syndrome, progressive cerebellar and cerebral

41 atrophy

42 Introduction

43 Pontocerebellar hypoplasia (PCH) is a group of neurodegenerative disorders with autosomal

44 recessive inheritance. Up to date 11 different subtypes have been described, with 17 causative

45 genes identified (van Dijk et al., 2018). Most of the subtypes are characterized by prenatal or

46 neonatal onset, global developmental delay and intellectual disability, microcephaly, hypoplasia

47 and variable atrophy of cerebellar cortex and/or brainstem. The specific neurological symptoms

48 and the severity of symptoms and brain loss vary between the subtypes (van Dijk et al., 2018).

49 Pontocerebellar hypoplasia type 6 (PCH6; MIM 611523) is a rare form of PCH first described in

50 2007 in three patients of a consanguineous Sephardic Jewish family (Edvardson et al., 2007). Since

51 then, altogether 32 patients in 18 families have been reported in the literature (for a detailed

52 summary of the patients and phenotypes, see Supplementary Table; Edvardson et al., 2007; Rankin

53 et al., 2010; Namavar et al., 2011; Glamuzina et al., 2012; Cassandrini et al., 2013; Kastrissianakis

54 et al., 2013; Joseph et al., 2014; Li et al., 2015; Lax et al., 2015; Nishri et al., 2016; Alkhateeb et al.,

55 2016; Ngoh et al., 2016; van Dijk et al., 2017; Luhl et al., 2016; Zhang et al., 2018). Most PCH6

56 patients present with neonatal onset, hypotonia, microcephaly, seizures, severe intellectual

57 disability with lack of developmental milestones and progressive atrophy of cerebral cortex,

58 cerebellum and pons. The majority show a respiratory chain enzyme deficiency and elevated

59 lactate levels in blood or cerebrospinal fluid (CSF). Indeed, PCH6 may be distinguished from the

60 other PCH subtypes, which are highly variable clinically and neuroradiologically, by the presence of

61 elevated lactate concentration (van Dijk et al., 2018).

62 PCH6 is caused by biallelic mutations in RARS2 , a nuclear gene that encodes the mitochondrial

63 arginine-tRNA synthetase enzyme (mtArgRS) (Edvardson et al., 2007). Aminoacyl-tRNA synthetases

64 play a crucial role in protein translation as they catalyze the specific attachment of an amino acid

65 (aminoacylation) to its cognate tRNA. MtArgRS participates in the synthesis of all 13 mitochondrial-

66 encoded proteins by charging of mitochondrial tRNA-Arg, thus being an integral part of

67 mitochondrial protein translation machinery, participating in generation of complexes of oxidative

68 phosphorylation system, except complex II, which has a fully nuclear origin (Ibba and Soll, 2000).

69 PCH6 shows clinically some resemblance to PEHO syndrome (Progressive Encephalopathy with

70 oedema, Hypsarrhythmia and Optic atrophy; MIM 260565), characterized by neonatal hypotonia,

71 profound psychomotor retardation, infantile spasms with hypsarrhythmia and atrophy of optic

72 disks (Salonen et al., 1991). Patients present with typical dysmorphic features, such as narrow

73 forehead, epicanthic folds, short nose and open mouth, and edema of the face and limbs (Somer,

74 1993). Neuroimaging findings include demyelination and progressive atrophy of the cerebellar

75 cortex, brainstem and optic nerves. In the cerebellum, the inner granular layer is nearly totally

76 absent and Purkinje cells are deformed and disaligned (Haltia and Somer, 1993).

77 PEHO syndrome is inherited autosomal recessively and was recently shown to be caused in Finnish

78 patients by a homozygous missense mutation c.92C>T; p.Leu31Ser in ZNHIT3 , a gene encoding zinc

79 finger HIT domain-containing protein 3 (Anttonen et al., 2017). PEHO syndrome is enriched in the

80 Finnish population with an estimated incidence of 1:74 000 (Somer, 1993) and approximately 40

81 diagnosed patients. In other populations it is very rare, with less than 25 reported patients (Field

82 et al., 2003; Caraballo et al., 2011; Alfadhel et al., 2011) and only one patient with compound

83 heterozygous mutations in ZNHIT3 reported so far (Öunap et al., 2019). In the literature, patients

84 with symptoms closely resembling PEHO syndrome are more commonly reported. The clinical

85 presentation of patients with PEHO-like features, like those with PCH, is similar to that of PEHO

86 syndrome, but optic atrophy and typical neuroradiologic findings are usually absent or there is no

87 progression (Field et al., 2003; Longman et al., 2003; Chitty et al., 1996). Several genes underlying

88 phenotypes resembling PEHO have been described (Rankin et al., 2010; Anttonen et al., 2015;

89 Gawlinski et al., 2016; Langlois et al., 2016; Nahorski et al., 2016; Flex et al., 2016; Miyake et al.,

90 2016; Zollo et al., 2017; Chitre et al., 2018).

91 We report a patient with the initial presenting features suggestive of PEHO syndrome with typical

92 dysmorphic features, epileptic spasms, optic atrophy and severe hypotonia, but in whom whole

93 genome sequencing revealed novel compound heterozygous mutations in RARS2.

94 Materials and methods

95 Patient and samples

96 The proband was clinically examined by B.C. in Antwerp and was referred to molecular genetic

97 analyses in Helsinki. DNA extracted from peripheral blood was obtained from the proband and

98 both parents. Primary fibroblast cultures from the proband were available for analyses of the gene

99 product.

100 An institutional review board at the Helsinki University Central Hospital approved the study. A

101 written informed consent was obtained from the parents.

102

103 Sequencing of ZNHIT3

104 The five coding exons of ZNHIT3 (NM_004773.3) were Sanger sequenced from genomic DNA of

105 the proband (primer sequences available upon request). Exon 1 covering the c.8C>T, p.Ser3Leu

106 variant was also sequenced in the parents.

107

108 Whole genome sequencing

109 Library preparation for the genomic DNA sample was performed using KAPA Library Preparation

110 Kit. The sample was sequenced in three lanes of an Illumina HiSeq2500 instrument with one lane

111 having paired-end 250-bp reads and two lanes paired-end 10-bp reads. Sequence read alignment

112 to human reference genome (GRCh37) and variant calling (Li et al., 2009) was done as described

113 earlier with minor modifications (Sulonen et al., 2011). Called variants were annotated using

114 ANNOVAR (Wang et al., 2010) and filtered using in-house scripts. DELLY (Rausch et al. 2012), which

115 assesses split-read alignments and paired-end read information to detect structural variants was

116 used to identify any copy number changes overlapping with the ZNHIT3 locus. Sanger sequencing

117 was performed from genomic DNA of the patient and the parents to validate the variants

118 identified by whole genome sequencing and to test segregation of the variants in the family.

119 Primer sequences are available upon request.

120

121 Sequencing of patient cDNA

122 Patient fibroblasts were harvested, total RNA extracted (RNeasy plus mini kit, QIAGEN) and

123 complementary DNA (cDNA) prepared (iScript cDNA synthesis kit, BioRad). Polymerase chain

124 reaction was performed using primers (sequences available upon request) binding to exons 8 and

125 14 of RARS2 and the resulting 600-bp product covering the positions of the mutations in exons 10

126 and 11 was sequenced using standard protocols.

127

128 Western blot analysis

129 Protein extracts for the detection of mtArgRS, COXII or GAPDH were prepared by lysing fibroblasts

130 in RIPA buffer (Cell Signaling Technology) containing protease inhibitors (Halt, Thermo Fisher

131 Scientific). After 10 min incubation on ice the samples were centrifuged at 14 000 g for 10 min (+4

132 ⁰C). Proteins were separated by SDS-PAGE and transferred onto membranes. After blocking with

133 5% milk in 0.1% TBS–Tween 20, the membranes were incubated with the corresponding primary

134 antibodies: rabbit anti-human mtArgRS (1:1000, Biorbyt, orb374171), rabbit anti-human COXII

135 (1:500, GeneTex, GTX62145) or rabbit anti-human GAPDH (Cell Signaling Technology, 14C10).

136 Reactive bands were detected using horseradish peroxidase-conjugated secondary antibodies

137 (goat anti-rabbit or goat anti-mouse, 1:10 000, Life Technologies). Blots were imaged using the ECL

138 western blotting substrate (Thermo Fisher Scientific) and Chemidoc XRS+ Molecular Imager (Bio-

139 Rad). Quantification of the band intensities was performed with the Image Lab Software (Bio-Rad).

140

141 Northern blot and aminoacylation assay

142 Total RNA was extracted from cultured fibroblasts using Trizol reagent (Thermo Fisher scientific)

143 according to the manufacturer’s instructions. To preserve the aminoacylation state the final RNA

144 pellet was re-suspended in 10mM NaOAc at pH 5.0. To investigate the aminoacylation status of mt-

145 tRNAs, 4μg of RNA was separated on long (16cm length) 6.5% polyacrylamide gel (19:1

146 acrylamide:bis-acrylamide) containing 8M urea in 0.1 NaOAc, pH 5.0. The fully deacylated tRNA

147 (dAc) was obtained by incubation of the control RNA at 75°C (pH 9.0) for 15 min. To determine mt-

148 tRNA Arg steady-state levels the samples were run on 10cm gel. Northern hybridization was

149 performed with ϒ-32P labeled oligonucleotide probes: 5’-GAGTCGAAATCATTCGTTTTG-3’ for the

150 mt-tRNA Arg and 5’- GTGGCTGATTTGCGTTCAGT-3’ for the mt-tRNA Ala . Radioactive signal was

151 detected by PhosphorImager plate using Typhoon scanner and quantified with the ImageQuant

152 v5.0 software (GE Healthcare).

153 Results

154 Clinical description

155 The essential clinical features in our patient are summarized in Supplementary Table. The patient

156 was the first child of non-consanguineous Belgian parents. Family history was unremarkable. He

157 was born at term after an uneventful pregnancy. Birth weight was 3.150 kg (-1 SD), length 50 cm (-

158 1 SD) and head circumference 35 cm (-0.5 SD). After birth slight hypothermia occurred, leading to

159 one day neonatal care, but otherwise physical examination was normal. Very early psychomotor

160 milestones were reported normal, but at the age of 2 to 3 months lack of social interaction, late

161 visual contact and mild hypotonia were noted. No further developmental milestones were

162 reached, he had no speech and showed no real social contact. The patient had no dysmorphic

163 signs at birth, but later presented with bitemporal narrowing, high palate, open mouth, full

164 cheeks, a tented upper lip (Fig. 1A) as well as mild edema of hands (Fig. 1B) and feet. Eye

165 examination showed no visual contact and a pale papilla on both eyes later progressing to optic

166 atrophy. Due to feeding difficulties the child was tube fed. An acquired microcephaly was noted

167 with occipitofrontal circumference (OFC) of 43 cm (-3.3 SD) at the age of 1 year and 46 cm (-3.7

168 SD) at the age of 3 years. At the last clinical follow-up with 9 years of age, he presented as a

169 bedridden child with profound intellectual disability, axial hypotonia, spastic quadriplegia and

170 significant seizure burden.

171 First convulsions were witnessed at the age of 6 weeks with lateralized clonic movements of the

172 face, followed by diminished consciousness and eye deviation to one side as well as bilateral clonic

173 movements of the body. It is unclear from the history whether these seizures were already

174 present from birth. Convulsions evolved into therapy-resistant epilepsy with varying seizure types:

175 complex focal seizures (with and without diminished consciousness) with myoclonic jerks and

176 laughing, rhythmic clonic movements of one or both limbs and long-lasting eye deviations with

177 nystagmus. The patient suffered from daily seizures several times a day with isolated myoclonic

178 spams and clusters in between.

179 EEG studies at the age of one to 3 months showed normal background activity without any

180 epileptic activity. Multifocal epileptic activity was seen from the age of 4 months and high voltage

181 slow background activity from the age of 5 months. The EEG did show some signs of

182 hypsarrhythmia and could, because lack of total desynchronization, be described as a modified

183 hypsarrhythmia. The last EEG recording, taken one day before the patient died, demonstrated a

184 picture of status epilepticus with continuous multifocal epileptic activity.

185 Magnetic resonance imaging (MRI) was performed at the ages of 4.5 months and 7 years. At 4.5

186 months (Fig. 1C,D), it showed severe cerebral atrophy, destruction of the thalami, and delayed

187 myelination, whereas the cerebellum appeared normal in size. At 7 years (Fig. 1E-G), the

188 cerebellar atrophy was prominent, and microcephaly masked some of the cerebral atrophy. The

189 pons was normal, and the myelination had reached almost a normal appearance.

190 Thorough metabolic investigations were unremarkable, with the exception of an intermittently

191 raised serum lactate up to 5.3 mmol/l (0.5-2 mmol/l) and an elevated lactate level in the CSF, up to

192 2.8 mmol/l (<2.5 mmol/l). No abnormalities were seen in the muscle biopsy.

193 Prior genetic investigations including karyotype and microarray came out normal and

194 mitochondrial DNA mutations were excluded.

195 The patient died at the age of nearly 12 years due to a respiratory infection.

196

197 Molecular findings: RARS2 mutations and their consequence

198 Given that the patient presented with symptoms overlapping with those reported in PEHO

199 syndrome, his DNA was first Sanger sequenced to identify variants in the coding regions and splice

200 sites of ZNHIT3 . A rare heterozygous c.8C>T, p.Ser3Leu (NM_004773.3) missense variant was

201 identified, but the patient did not have other ZNHIT3 coding sequence variants. To identify any

202 non-coding variants in ZNHIT3 locus, the patient was whole genome sequenced. Analysis for rare

203 sequence variants in intronic or UTR regions of ZNHIT3 , or up- or downstream to ZNHIT3 did not

204 identify a second variant. No copy number changes overlapping with the ZNHIT3 locus was

205 identified.

206 Analysis of the whole genome data was then expanded to all protein coding regions of the

207 genome and splice sites. Whole genome sequence data was produced with mean sequencing

208 coverage of 24.48x, and 98.2%, 95.7% and 74.2% of the genome was covered at least 5x, 10x and

209 20x, respectively. Analysis of the coding regions from the genome sequence data focused on rare

210 heterozygous and potentially biallelic variants in established disease genes. Analysis of rare

211 heterozygous variants did not yield any likely candidates explaining the patient’s disease. Analysis

212 of rare biallelic variants revealed two heterozygous variants in RARS2 (NM_020320.3; Fig. 2A and

213 B; https://databases.lovd.nl/shared/individuals/00234052), a one-bp deletion in exon 10 causing a

214 frameshift and premature termination of translation 16 codons downstream (c.795delA,

215 p.Glu265Aspfs*16) and a missense variant, c.961C>T, p.Leu321Phe, in exon 11. There is one

216 heterozygous carrier for the c.961C>T, p.Leu321Phe variant in the gnomAD (Lek et al., 2016)

217 database (v. 2.0; allele frequency 0.000004), whereas the frameshift variant is absent from the

218 database. The leucine at position 321, located in the catalytic domain of RARS2, is highly

219 conserved (Fig. 2B). In silico tools SIFT, PolyPhen-2 and MutationTaster predict the c.961C>T,

220 p.Leu321Phe substitution as deleterious. Sanger sequencing confirmed compound heterozygosity

221 of the two mutations in the patient: the c.795delA frameshift mutation was inherited from the

222 mother and the c.961C>T missense mutation from the father (Fig. 2A).

223 The consequence of the RARS2 variants was studied on mRNA level in skin fibroblasts of the

224 patient. The frameshift variant in exon 10 resulting in a premature termination codon is predicted

225 to be subjected to nonsense-mediated mRNA decay (NMD) and degradation of the transcript

226 derived from the maternal allele. Indeed, sequencing of RARS2 cDNA revealed that at position

227 c.961 only the paternal C>T variant was present (Fig. 2C). Western blot analysis of patient

228 fibroblasts revealed that the mtArgRS protein level was reduced to about 50 % of control level (Fig.

229 3A). Northern blot analysis of total RNA from fibroblasts suggested that the steady-state level of

230 mitochondrial tRNA Arg when compared to mitochondrial tRNA Ala may be decreased in patient

231 fibroblasts (Fig. 3B). In patient and control fibroblasts, aminoacylation analysis showed the

232 presence of only aminoacylated mt-tRNA Arg , whereas deacylated mt-tRNA Arg was not detected (Fig.

233 3C). This finding is in agreement with the previous observation (Edvardson et al., 2007 ), suggesting

234 that in cultured human fibroblasts uncharged mt-tRNA Arg is not stable.

235 Discussion

236 We describe a patient compound heterozygous for two novel pathogenic variants in RARS2, the

237 gene associated with PCH6. The high conservation of the affected Leu321, the predicted

238 deleteriousness of the Leu321Phe substitution combined with degradation of the transcript

239 derived from the allele with the frameshift variant strongly suggest that these variants are the

240 underlying cause for PCH6 in the patient.

241 The role of RARS2 in pontocerebellar hypoplasia is not fully understood with no clear genotype-

242 phenotype correlations. It is though likely that the severity of the disease is dependent of the

243 amount of remaining aminoacylation activity (Konovalova and Tyynismaa, 2013). mtArgRS has a

244 fundamental function in mitochondrial protein synthesis, so total loss-of-function mutations are

245 likely to be lethal. Compatible with this notion, mice homozygous for a knock-out allele of Rars2

246 are embryonic lethal (International Mouse Phenotyping Consortium;

247 http://www.mousephenotype.org/data/genes/MGI:1923596#section-associations). Considering

248 the markedly reduced expression from the frameshift allele, the missense mutant allele is likely to

249 retain some mtArgRS activity in our patient. It has been suggested that due to the leaky nature of

250 the mutations, small amounts of protein synthesis is possible in most tissues, but in high energy

251 demanding cells, such as neurons, the reduced aminoacylation is not sufficient thus causing the

252 symptoms of the disease (Edvardson et al., 2007). Low enzyme activity affects the development of

253 the central nervous system already in utero as demonstrated by abnormal brain MRI findings in

254 the neonatal period (e.g. Edvardson et al., 2007; Joseph et al., 2014; Lax et al., 2015). It is also

255 possible that the reduced aminoacylation of tRNA-Arg has bigger effect on specific neuronal types

256 that causes the alterations in brain typical for PCH6. There is also evidence of particular uncharged

257 tRNAs and amino acids working as potential signaling molecules (Dong et al., 2000; Wolfson et al.,

258 2016). Mitochondrial tRNA synthetases may also have non-canonical functions, similarly to their

259 cytosolic counterparts, in addition to their housekeeping function in protein synthesis, and these

260 may contribute to the pathomechanisms. For example, mtArgRS was recently found to have a

261 specific sub-mitochondrial localization in the membrane, which suggests that it also could have

262 alternative functions (Gonzáles-Serrano et al., 2018 ). Regardless of the reason, this high tissue

263 specificity makes functional studies of the disease mechanism challenging.

264 Including the present patient, 33 patients with PCH6 in 19 families have been described

265 (Supplementary Table). An overview of the key clinical features in the patients is presented in Table

266 1. Most patients were normal at birth but presented with variable symptoms at early age (hours to

267 9 months). First presenting features included hypotonia in 15/33 patients and seizures in 16/33

268 patients. Other early symptoms were poor feeding, lethargy and apneic episodes. All patients were

269 reported to have global developmental delay and the majority presented seizures, the onset

270 varying from 9 hours to several months. Most seizures were intractable myoclonic or tonic-clonic

271 seizures, either focal, or multifocal or generalized. Other common features in the patients include

272 progressive microcephaly, atrophy of cerebellum and cerebrum, as well as elevated lactate levels

273 in blood or CSF. Notably, atrophy of pons was reported to be present in only 12 out of the 25

274 patients with reported MRI findings, indicating that pons can be normal in PCH6 (Nishri et al.,

275 2016). The phenotype in our patient is similar to that of previously published patients, and

276 presents with all features listed in Table 1, except atrophy of the pons. Of note, as in at least three

277 published patients (Ngoh et al., 2016; Zhang et al., 2018; Luhl et al., 2016), the serum lactate levels

278 in our patient were intermittently raised.

279 Compatible with a previous report (Rankin et al., 2010), the initial clinical features in our patient

280 including severe intellectual disability, epilepsy, optic atrophy, hypotonia, acquired microcephaly,

281 mild edema of hands and feet, and dysmorphic features pointed to PEHO syndrome. Although the

282 dysmorphic features raised the suspicion of the PEHO syndrome, they may, however, be non-

283 specific, as many of the dysmorphic facial features are associated with developing microcephaly,

284 extreme floppiness, and edema (Somer, 1993). Contrary to findings in our patient, patients with

285 PEHO syndrome do not show elevated lactate levels in blood or CSF and usually present with

286 neonatal hypotonia (Anttonen et al., 2017). Importantly, the MRI findings in our patient (Fig. 1C-G)

287 were not typical for PEHO syndrome. The supratentorial atrophy was more severe than in a typical

288 PEHO patient. Moreover, the myelination was not delayed to the degree seen in PEHO patients.

289 Characteristic MRI findings including progressive cerebellar atrophy and dysmyelination are

290 essential diagnostic criteria for PEHO syndrome (Anttonen et al., 2017). These typical findings are

291 often disregarded when suggesting a clinical PEHO diagnosis.

292

293

294 Acknowledgements

295 We thank the family for their contribution to this study. We thank the Genome Aggregation

296 Database (gnomAD) and the groups that provided exome and genome variant data to this

297 resource. A full list of contributing groups can be found

298 at http://gnomad.broadinstitute.org/about. This study was funded by the Folkhälsan Research

299 Foundation.

300

301

302 Accession numbers

303 https://databases.lovd.nl/shared/individuals/00234052

304 Figure Titles and Legends

305 Figure 1. Phenotypic features in the patient.

306 A) Facial features of the patient at 7 years of age. Note the open mouth, full cheeks, a tented

307 upper lip and bitemporal narrowing. B) The hand shows edema. C) In a sagittal T1-weighted cranial

308 magnetic resonance image at the age of 4.5 months cerebellum (arrowhead) and pons (arrow)

309 appear normal in size. D) T2-weighted axial image at 4.5 months shows cerebral atrophy.

310 E & F) T2-weighted images of the patient at 7 years of age show microcephaly and widespread

311 cerebral atrophy as well as severe cerebellar atrophy (arrowhead in E) with widened cerebellar

312 sulci ( F). The pons (arrow in E) as well as the myelination appear normal. G) T2-axial slices at 7

313 years also show atrophy and signal increase of the thalami (open arrowheads).

314

315 Figure 2. Two novel PCH6-associated mutations in the RARS2 gene.

316 A) Sanger sequencing chromatograms of the proband’s (P) and the parents’ genomic DNA showing

317 the c.795delA variant inherited from the mother (M) and the c.961C>T variant inherited from the

318 father (F). Positions of variants are indicated with arrowheads. B) A schematic picture of the exon-

319 intron structure of RARS2 and the domain structure of the encoded protein (modified from

320 Gonzáles-Serrano et al., 2018) showing the locations of the identified mutations and high

321 conservation of the leucine at position 321 affected by the missense substitution. C) Sanger

322 sequencing chromatograms of the proband’s cDNA showing only the paternal c.961C>T variant

323 (arrowhead) in exon 11 suggesting that the transcript derived from the maternal allele is

324 degraded. 11F denotes forward orientation sequence and 11R reverse orientation

325

326 Figure 3. Western blot, northern blot and aminoacylation analysis of the patient fibroblasts.

327 A) Steady-state level of mtArgRS protein in patient (P) and control fibroblasts (C1, C2) detected by

328 Western blot. Quantification of the Western blot analysis is shown in the right panel. GAPDH was

329 detected as protein loading control. Data are presented as mean ± SD. B) Northern blot analysis of

330 mt-tRNA Arg levels in patient (P) and control (C1, C2) fibroblasts. Quantification of the northern blot

331 analysis is shown in the lower panel. Mitochondrial tRNA Ala was detected as a loading control.

332 C) Aminoacylation assay of mt-tRNA Arg in control (C1, C2) and patient (P) fibroblasts. Mitochondrial

333 tRNA Ala was detected as a loading control. dAC denotes the fully deacylated control tRNA.

334 Experiments in B and C were carried out only once.

335 Table 1. Overview of clinical features in published PCH6 patients 336

Feature n/na

Global developmental delay 33/33 Epileptic seizures 24/24 Microcephaly 20/27 MRI findings Atrophy of cerebellum 22/25 Atrophy of pons 12/25 Atrophy of cerebrum 18/25 Elevated lactate level in blood or CSF 19/23 Reduced respiratory chain enzyme activity 10/19 Feeding difficulties 17/18 Dysmorphic features 6/8

337 338 CSF – cerebrospinal fluid 339 a 340 The features are variably reported in the patients.

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539 Supplemental Data

540 Supplementary Table: Phenotypic features in published PCH6 patients